Project summary Mycobacterium tuberculosis (Mtb) is the leading cause of death due to an infectious organism. However, approximately 90% of individuals who are infected remain disease free and maintain a latent infection. The mechanisms by which some individuals are protected while others succumb remains an open question. Clearly diseases affecting immune function such as AIDS or environmental factors that promote lung damage such as smoking are major risk factors. Nevertheless, we still lack a comprehensive understanding of what immune factors are required for controlling an Mtb infection. Our published studies suggest that exosomes, small membrane vesicles released from most nucleated cells, may be playing a previously unidentified role in regulating an immune response to an Mtb infection. We have found that exosomes released from Mtb-infected macrophages contain mycobacterial components and can stimulate both an innate and acquired immune response in vitro and in vivo. Recent studies have identified Mtb RNA as one of the components present in these exosomes. This interesting and unexpected finding suggest that host RNA sensors may be activated during the course of a mycobacterial infection. Surprisingly we know little in regards to the importance of bacterial RNA in stimulating a host immune response. In contrast, numerous studies have shown viral RNA to be major drivers of a cellular response through activation of RNA sensors such as TLR3 and Rig-1. Therefore a better understanding of how mycobacterial RNA is packaged into exosomes and how these exosomes function in host immunity will open up new areas of investigation into bacterial RNA as immune modulators as well as gain further insight into the role of exosomes in the host response to an Mtb infection. The importance of Mtb RNA is supported by our preliminary data which shows that exosomes released from infected but not from uninfected macrophages can induce IFN-? production in nave macrophages and do so in an Mtb RNA dependent manner. We hypothesize that Mtb RNA is secreted during an active infection and functions through exosomes as an important driver of the host immune response. Therefore, we propose in Specific Aim 1 to characterize the mechanism by which Mtb RNA is trafficked to exosomes as well as define the composition of the Mtb RNA within the exosomes. While in specific aim 2 we will use in vitro and in vivo studies to characterize the function of the Mtb exosomal RNA. Information garnered from these studies will not only provide new information on the mechanism of immune regulation during an Mtb infection but also provide potential new targets for tuberculosis diagnostics in the form of exosomal RNA.